CA2185394C

CA2185394C - Biotinderivate

Info

Publication number: CA2185394C
Application number: CA002185394A
Authority: CA
Inventors: Alfred Jonczyk; Simon Goodman; Beate Diefenbach; Horst Kessler; Dirk Finsinger
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 1995-09-14
Filing date: 1996-09-12
Publication date: 2007-05-01
Anticipated expiration: 2016-09-12
Also published as: CZ262996A3; UA41981C2; AU6551896A; TW517063B; EP0771818A3; EP0771818A2; HU9602223D0; DE59608995D1; SK283129B6; SK115696A3; ATE215558T1; NO963851L; RU2171807C2; PL316069A1; CN1168738C; BR9603741A; JPH09124692A; JP4127325B2; ES2174007T3; DE19534016A1

Abstract

Biotin compounds of the formula I (see formula I) is which Q is absent, -NH-(CH2)n-CO or -NH-(CH2)n-NH- and R1 is X-Arg-Gly-Asp-Y, A-Cys(R2)-B-U or cyclo-(Arg-Gly-Asp-Z), where Z is bonded in the side chain to Q or, if Q is absent, to biotin, and A, B, U, X, Y, Z and n have the meanings stated is Claim 1, and their salts can be used as integrin inhibitors, in particular for the prophylaxis and treatment of disorders of the circulation, for thrombosis, myocardial infarct, coronary heart diseases, arteriosclerosis, angiogenic disorders and is tumour therapy.

Description

Biotinderivate The invention relates to biotin compounds of the formula I
"a ~o , ~N

R~
Q
in which Q 1.8 absent, -NH- (CHZ) a-C~- Or -N8- (CH2) n-NH-, R1 is X-Arg-Gly-Asp-Y, A-Cys(R2)-H-U or cyclo-(Arg-Gly-Asp-Z), where Z is bonded in the side chain to Q or, if Q is absent, to biotin, X and Y are each, independently of one another, an amino-acid residue or a di-, tri-, tetra- or pentapeptide residue, where the amino acids are selected, independently of one another, from a group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, 4-Hal-Phe, His, homo-Phe, IIe, Leu, Lys, Met, Nle, Phe, Phg, Pro, Ser, Thr, Trp, Tyr or Val, and the said amino acids can also be derivatized, A is absent, Asp or a peptide fragment selected from a group consisting of Ala-Asp, Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Thr-Ala-Asn, Lys-Thr-Gly-Asp, Lys-Ala-Ala-Asp, Arg-Thr-Ala-Asp, Ser-Ala-Asp, Gln-Ser-Ala-Asp, Glp-Ser-Ala-Asp, Gly-Lys-Thr-Ala-Asp, Asn-Gly-Lys-Thr-Ala-Asp, Ile-Ser-Ala-Gly, Arg-Ser-Ala-Gly, Cys-Asn-Gly-Lys-Thr-Ala-Asp, _ 2 _ 2185394 -Tyr-Cys-ASn-Gly-Lys-Thr-Ala-Asp, Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Gly-Lys-Thr-Cys(Trt)-Asp, Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Ala-Asp, B is absent, OH, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Orn, Phe, 4-Hal-Phe, Pro, Ser, Thr, Trp, Tyr, Val or an N-methylated derivative of the said amino-acid residues, or a peptide fragment selected from a group consisting of Pro-Arg, Pro-Arg-Asn, Pro-Arg-ASn-Pro, Pro-Arg-Asn-Pro-His, Pro-Arg-Asn-Pro-His-Lys, Pro-Arg-Asn-Pro-His-Lys-Gly, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala, Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr, ao where, if R1 is A-Cys(R2)-B-U, only one of the radicals A or B can be absent, RZ is H, alkyl with 1-6 C atoms, Trt, Dpm or Bzl, U is OH, OR9, NHz, NHR9 or N(R9)z Z is in each case, independently of one another, an amino-acid residue or a di-, tri- or tetra-peptide residue, where the amino acids thereof can also be selected, independently of one another, from a group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val or M, where the said amino acids can also be deriva-tiled, and the amino-acid residues can be linked together in the manner of a peptide via the a-amino and a-carboxyl groups, with M always being present, M is NH(RB)-CH(R;)-COOH, R3 is -R5-R~, -R6-R~. -R~-Rt.
R4 is OH, NHs, SH or COOH, RS is alkylene with 1-6 C atoms, R6 is alkylenephenyl with 7-14 C atoms, R' is alkylenephenylalkylene with 8-14 C atoms.
RB is H, alkyl with 1-6 C atoms or alkylenephenyl with 7-12 C atoms, R9 is alkyl with 1-6 C atoms, Hal is F, C1, Br or I and n is 1, 2, 3, 4. 5, 6, 7, 8, 9 or 10, where both the D and the L forms are included if radicals of optically active amino acids and amino-acid derivatives are present, and the salts thereof.
Similar compounds of biotinylated peptides are described, for example, in WO 9415956 (biotinylated endothelia receptor antagonists), in WO 9413313 (bio-tinylated LHRH antagonists) or WO 9418325 (biotinylated necrosis factor).
The biotinylatioa of peptides during solid-phase syn-thesis on a resin for the purpose of improving the possibility of purification is described by T.J. Lobl et al. in Anal. Biochem. 170, 502 (1988). Similar com-pounds of cyclic and linear peptides are disclosed in = ~~ - 4 - 218594 DE 43 10 643, DE 43 36 758, EP 0 406 428 and WO 89/05150.
The invention was based on the object of finding novel compounds with valuable properties, especially those which can be used to produce pharmaceuticals.
It has been found that the compounds of the formula I and their-salts have, while being well tole-rated, very valuable pharmacological properties. In particular, they sat as integrin inhibitors, and they inhibit in particular the interactions of the a", (33 or [35 integrin receptors with ligands, such as, for example, the binding of fibrinogen to the (i3 integrin receptor. The compounds show particular activity in the case of the integrins a~~i3, axis, a=~(33 and a"[31, ao[36 and a~,(3a. This effect can be detected, for example, by the method described by J.W. Smith et al. in J. Biol. Chem.
265, 12267-12271 (1990).
The dependence of the beginning of angiogenesis on the interaction between vascular integrins and extra cellular-matrix proteins is described by P.C. Brooks, R.A. Clark and D.A. Cheresh in Science ~~~, 569-71 (1994).
The possibility of inhibiting this interaction and thus of initiating apoptosis (programmed cell death) of angiogenic vascular cells by a cyclic peptide is described by P.C. Brooks, A.M. Montgomery, M. Rosenfeld, R.A. Reisfeld, T.-Iiu, G. Rlier and D.A. Cheresh in Cell ZQ, 1157-64 (1994).
Compounds of -the formula I which block the interaction of integrin receptors and ligands, such as, for example, of fibrinogen on the fibrinogen receptor (glycoprotein IIb/IIIa), prevent as GPIIb/IIIa antago nists the spread of tumour cells by metastasis. This is proved by the following observations:
The spread of tumour cells from a local tumour into the vascular system takes place by the formation of microaggregates (microthrombi) by interaction of the tumour cells With blood platelets. The tumour cells are - ~~ - 5 - 218594 - shielded by the protection in the microaggregate and are not recognized by the cells of the immune system.
The microaggregates may become attached to vessel walls, which facilitates further penetration of tumour cells into the tissue. Since the formation of microthrombi is mediated by fibrinogen binding to the fibrinogen receptors on activated blood platelets, the GPIIa/IIIb antagonists can be regarded as effective inhibitors of metastasis.
The compounds of the formula I can be employed as pharmaceutical active substances in human and veterinary medicine, in particular for the prophylaxis and/or therapy of- thrombosis, myocardial infarct, arteriosclerosis, inflammations, stroke, angina pectoris, oncoses, osteolytic diseases such as oateo-poroais, pathologically angiogenic disorders such as, for example, inflammations, ophthalmological disorders, diabetic retinopathy, macular degeneration, myopia, ocular histoplasmosis, rheumatoid arthritis, osteoarthritis, rubeotic glaucoma, ulcerative colitis, Crohn's disease, atherosclerosis, psoriasis, restenosis after angioplasty, viral infection, bacterial infection, fungal infection, for acute kidney failure and for wound healing to support the healing processes.
The compounds of the formula I can be employed as antimicrohially active substances in operations where biomaterials, implants, catheters or cardiac pacemakers are inserted. In this case, they act as antiseptics. The efficacy of the antimicrobial activity can be demonstrated by the method described by P. Valentin-Weigund et al. in Infection and Immunity, 2851-2855 (1988).
Since the compounds of the formula I are inhibitors of fibrinogen binding and thus ligands of the fibrinogen receptors on blood platelets, they can be used as diagnostic aids for detecting and locating thrombi in the vascular system in vivo, because the biotinyl radical is a W-detectable radical.

~

The compounds of the formula I can, as inhibitors of fibrinogen binding, also be used as effective aids for studying the metabolism of blood platelets in various stages of activation or of intra-cellular signal mechanisms of the fibrinogen receptor.
The detectable unit of the °biotin label° makes it possible to investigate the said mechanisms after binding to the receptor.
The abbreviations of amino-acid residues employed hereinbefore and hereinafter represent the radicals of the following amino acids:
Abu 4-Aminobutyric acid Aha 6-Aminohexanoic acid, 6-aminocaproic acid Ala Alanine Asn Asparagine Asp Aspartic acid Arg Arginine Cys Cysteine Dab 2,4-Diaminobutyric acid Dap 2,3-Diaminopropionic acid Gln Glutamine Glp Pyroglutamic acid Glu Glutamic acid Gly Glycine His Histidine homo-Phe homo-Phenylalanine Ile Isoleucine Leu Leucine Lys Lysine Meth Methionine Nle Norleucine Orn Ornithine Phe Phenylalanine Phg Phenylglycine 4-Hal-Phe 4-Halo-phenylalanine Pro Proline Ser Seriae Thr Threonine Trp Tryptophan Tyr Tyrosine Val Valine and H H
N
S ~O
Hit _ a Further meanings are the following:
BOC tert-Butoxycarbonyl CHZ Benzyloxycarbonyl DCCI Dicyclohexylcarbodiimide DMF Dimethylformamide Et Ethyl F~noc 9-Fluorenylmethoxycarbonyl HOHt 1-Hydroxybenzotriazole Me Methyl .

MBHA 4-Methylbenzhydrylamine Mtr 4-Methoxy-2,3.6-trimethyiphenylsulfonyl OBut tert-Butyl eater OMe Methyl ester OEt Ethyl ester POA Phenoxyacetyl TFA Trifluoroacetic acid Trt Trityl (triphenylmethyl).

Whea the abovementioned amino acids can occur in several enantiomericv forms, all these forms, and mixtures thereof (for example the D and the L forms), are also included hereiabefore and hereinafter, for example as constituent of the comp4unds of the formula I.
Further-more, the amino acids can be provided~with appropriate protective groups which are known per se, for example as constituent of compounds of the formula I.

_ g _ The compounds according to the invention also include so-called prodrug derivatives, that ie to say compounds of the formula I which have been modified, for example, with alkyl or acyl groups, sugars or oligopeptides and which are rapidly cleaved in the body to the active compounds according to the invention.
The invention furthermore relates to a process for the preparation of compounds of the formula I
according to Claim 1, and the salts thereof, characterized in that (a) a compound of the formula II

in which Q and R1 have the meaning stated in Claim 1, is reacted in an acylation reaction with a compound of the formula III
N
H
H O ' L
in which L is C1, Hr, I or a free or reactively functionally modified OH group, or b) a compound of the formula IV

264x4-384.
_ g _ in which R1 has the meaaing stated in Claim 1, is reacted in an acylation reaction with a compound of the formula V
s ~o H V
.' O
Q~L
in which Q has the meaning stated in Claim 1, and L is H, C1, Br, I or a free or reactively functionally modified 08 group, or c~ they are liberated from one of their functional derivatives by treatment with a solvolysing or hydrogenolysiag agent, , and/or in that a basic or acidic compound of the formula I is converted by treatment with sa acid or base into one of the salts thereof.
8ereinbefore and hereinafter, the radicals Q, Rl and L have the meanings stated for formulae I, II aad III unless expressly stated otherwise.
In the above formulae, alkyl is preferably methyl, ethyl, isopropyl~or tent-butyl.
Alkylene is preferably methylene, ethylene, propylene, butylene. pentylene or hexylene.
Alkylenepheayl is preferably benzyl or phenethyl.
Alkylenepheaylalkyleae is preferably 4-methyleaebeazyl or 4-ethylenebenzyl.

- 1° - 2 ~ 85394 The radical -R6-R4 is preferably 2-, 3- or 4-hydroxybenzyl, 2-, 3- or 4-aminobenzyl, 2-, 3- or 4-mercaptobenzyl, 2-, 3- or 4-carboxybenzyl, further-more preferably 2-, 3- or 4-hydroxyphenethyl, 2-, 3- or 4-aminophenethyl, 2-, 3- or 4-mercaptophenethyl or 2-, 3- or 4-carboxypheaethyl.
Q is preferably 6-aminohexanoic acid (6-amino-caproic acid) or is abseat.
M is preferably Dap, Ser, Cys, Asp, D-Asp, Dab, homoserine, homocysteine, Glu, D-Glu, Thr, Orn, Lys, D-Lys, 4-aminomethyl-Phe or 4-aminomethyl-D-Phe.
X is preferably Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, furthermore preferably Lys-Gly, Lys-Ala, Lys-~i-Ala, Tyr-Gly, Tyr-Ala, Tyr-(3-Ala, Phe-Gly, Phe Ala, Phe-(3-Ala, Tyr-Gly-Gly, Phe-Giy-Gly, Lys-Gly-Gly, Tyr-Giy Ala, Phe-Gty-Ala, Lys-Gty-Aia, Arg-Giy-Asp, Lys-Gly-Gly-Gly, Tyr-Giy-Gly-Gly, Phe-Gly-Gly-Gly, Lys-Gly-Gly-Ala, Tyr-Gty-Gty-Ala, Phe-Gfy-Gly-Ala, Lys-Giy-Giy-(3-Ata, Tyr-Gly-Gty-(i-Ala, Phe-Giy-Giy-[3-Ala,furthermore also Lys-Giy-Gly-Gly-Gly, Tyr-Giy-Gly-Giy-Gly, Phe-Gly-Giy-Gly-Gly, Lys-Gly-G(y-A!a-Gly, Tyr-Giy-Giy-Ata-Giy, Phe-Gly-G!y-Ala-Gly, Lys-Gly-Gly-(3-Ala-Gly, Tyr-Gly-Gly-E3-Ala-Gly or Phe-Gly-Gly-(3-Ala-Gly.
Y is preferably Ala, Asa, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, furthermore preferably Tyr-Ala, Tyr-Asn, Tyr-Asp, Tyr-Arg, Tyr-Cys, Tyr-Gln, Tyr-Glu, Tyr-Gly, Tyr-His, Tyr-Ile, Tyr-Leu, Tyr-Lys, Try-Met, Tyr-Phe, Tyr-Pro, Tyr-Ser, - li. - 2185394 Tyr-Thr, Tyr-Trp, Tyr-Tyr,Tyr-Val, Phe-Ala, Phe-Asn, Phe Asp, Phe-Arg, Phe-Cys, Phe-Gin, Phe-Glu, Phe-Gly, Phe-His, Phe-Ile, Phe-Leu, Phe-Lys, Phe-Met, Phe-Phe, Phe-Pro, Phe-Ser, Phe-Thr, Phe-Trp, Phe-Tyr, Phe-Val, Trp Ala, Trp-Asn, Trp-Asp, Trp-Arg, Trp-Cys, Trp-Gln, Trp-Glu, Trp-Gly, Trp-His, Trp-Ile, Trp-Leu, Trp-Lys, Trp-Met, Trp-Phe, Trp-Pro, Trp-Ser, Trp-Thr, Trp-Trp, Trp-Tyr, Trp-Val, Asp Ala, Asp-Asn, ~P-ASP, ~P-~9, ~P-CYs. AsP-Gln, Asp-Glu, Asp-Gly, AsP-His, Asp-Ile, Asp-Leu, Asp-Lys, Asp-Met, Asp-Phe, Asp-Pro, Asp-Ser, Asp-Thr, Asp-Trp, Asp-Tyr, Asp-Vat, Ser-Pro-Lys, Tyr-Pro-Lys, Phe-Pro-Lys, Trp-Pro-Lys, Asp-Pro-Lys, Ser-Gly-Lys, Tyr-Gly-Lys, Phe-Gly-Lys, Trp-Gly-Lys, Asp-Giy-Lys, Ser-Ala-Lys, Tyr-Ala-Lys, Phe Ala-Lys, Trp-A!a-Lys, Asp-Ata-Lys, Ser-Pro-Ala, Ser-Leu-Lys, Tyr-Leu-Lys, Phe-Leu-Lys, Trp-Leu-Lys, Asp-Leu-Lys, Ser-Ile-Lys, Tyr-Ile-Lys, Phe-ile-Lys, Trp-Ile-Lys, Asp-Ile-Lys, Ser-Pro-Aia-Ser, Tyr-Pro-Ala-Ser, Phe-Pro-Ala-Ser, Trp-Pro-Ala-Ser, Asp-Pro-Ala-Ser, Ser-Gty Ala-Ser, Tyr-Gty-Ala-Ser, Phe-Gly-Ala-Ser, Trp-Gly Ala-Ser, Asp-Gly Ala-Ser, Ser Ala Ala-Ser, Tyr-Afa Ala-Ser, Phe-Ala-Ala-Ser, Ttp Ata-Ala-Ser, Asp-Ala-Ala-Ser, Ser-Vat Ala-Sec, Tyr Val-Afa-Ser, Phe-Vat-Ata-Ser, Trp Val-Ala-Ser, Asp-Val-Ala-Ser, Ser-Leu Ala-Ser, Tyr-Leu-Ala-Ser, Phe-Leu-Ala-Ser, Trp-Leu-Afa-Ser, Asp-Leu Ala-Ser, Ser-Ile lUa-Ser, Tyr-tle Ala-Ser, Phe-Ile-Ala-Ser, Trp-ile Ala-Ser, Asp-tle Ata-Ser, further-more also Ser-Pro-Ata-Ser-Ser,Tyr-Pro Ala-Ser-Ser, Phe-Pro Ala-Ser-Ser, Trp-Pro-Ala-Ser-Ser, Asp-Pro Ala-Ser-Ser, Ser-Gly-A!a-Ser-Ser, Tyr-Gly-Ala-Ser-Ser, Phe-Gly-Ata-Ser-Ser, Trp-Gly-Ala-Ser-Ser, Asp-Giy-Ala-Ser-Ser, Ser-Als-Ala-Ser-Ser, Tyr-Afa-Ala-Ser-Ser, Phe-Ala-Ala-Ser-Ser, Trp Ala Ala-Ser-Ser, Asp Ala-Ala-Ser-Ser, Ser-Val Ala-Sec-Ser, Tyr Val Ala-Ser-Ser, Phe-Val-Ala-Ser-Ser, Trp Val-Ata-Ser-Ser, Asp-Va!-Ata-Ser-Ser, Ser-Leu Ala-Ser-Ser, Tyr-Leu Ala-Ser-Ser, Phe-Leu-Ata-Ser-Ser, Trp-Leu Ala-Ser-Ser, Asp-Leu-Ala-Ser-Ser, Ser-Ile Hla-Ser-Ser, Tyr-Ile AIaSer Ser, Phe-Ile Ala-Ser-Ser, Trp-Ile AIa-Ser-Ser or Asp-Ile-Ala-Ser-Ser.
The amino acids and amino-acid residues mentioned in the meanings for X, Y and Z can also be derivatized, with the N-methyl, N-ethyl, N-propyl, N-benzyl or Ca-methyl derivatives being preferred.
Additionally preferred are derivatives of Asp and Glu, in particular the methyl, ethyl, propyl, butyl, tert-butyl, neopentyl or benzyl esters of the side-chain carboxyl groups, furthermore also derivatives of Arg which can be substituted on the -NH-C(=NH)-NHz group by an acetyl, benaoyl, methoxycarbonyl or ethoxycarbonyl radical.
Furthermore, the amino acids and amino-acid residues mentioned in the meanings for X and Y can be provided with appropriate protective groups known per se.
Z is preferably M, furthermore preferably D-Phe-M, D-Trp-M, D-Tyr-M, D-Phe-Lys, D-Phe-D-Lys, D-Trp-Lys, D-Trp-D-Lys, D-Tyr-Lys, D-Tyr-D-Lys, D-Phe-Orn, D-Phe-Dab, D-Phe-Dap, D-Phe-D-Orn, D-Phe-D-Dab, D-Phe-D-Dap, D-Phe-4-aminomethyl-Phe, D-Phe-4-aminomethyl-D-Phe, D-Trp-4-aminomethyl-Phe, D-Trp-4-aminomethyl-D-Phe, D-Tyr-4-aminomethyl-Phe, D-Tyr-4-aminomethyl-D-Phe, D-Phe-Asp, D-Phe-D-Asp, D-Trp-Asp, D-Trp-D-Asp, D-Tyr-Asp, D-Tyr-D-Asp, D-Phe-Cys, D-Phe-D-Cys, D-Trp-Cys, D-Trp-D-Cys, D-Tyr-Cys, D-Tyr-D-Cys, Phe-D-Lys, Trp-D-Lys, Tyr-D-Lys, Phe-Orn, Phe-Dab, Phe-Dap, Trp-Orn, Trp-Dab, Trp-Dap, Tyr-Ora, Tyr-Dab, Tyr-Dap, Phe-4-aminomethyl-D-Phe, Trp-4-aminomethyl-D-Phe, Tyr-4-aminomethyl-D-Phe, Phe-D-Asp, Trp-D-Asp, Tyr-D-Asp, Phe-D-Cys, Trp-D-Cys, Tyr-D-Cys, D-Phe-Lys-GIy, D-Phe-M-Gly, D-Trp-Lys-Gly, D-Trp-M-Gly, D-Tyr-Lys-Gly, D-Tyr-M-Gly, D-Phe-Val-Lys, D-Phe-Gly-Lys, D-Phe-Ala-Lys, D-Phe-Ile-Lys, D-Phe-Leu-Lys, D-Trp-Val-Lys, D-Trp-Gly-Lys, D-Trp-Ala-Lys, D-Trp-Ile-Lys, D-Trp-Leu-Lys, D-Tyr-Val-Lys, D-Tyr-Gly-Lys, D-Tyr-Ala-Lys, D-Tyr-Ile-Lys, D-Tyr-Leu-Lys, furthermore also M-Pro-Ala-Ser-Ser.
The compounds of the formula I may have one or more chiral centres and therefore occur in various stereoisomeric forms. Formula I embraces all these forms.
Accordingly, the invention relates in particular to those compounds of the formula I in which at least one of the said radicals has oae of the meanings indicated above as preferred. Some preferred groups of compounds can be represented by the following part-formulae Ia to If which correspond to the formula I and - is - 2$5394 in which the undefined radicals have the meanings stated for formula I, but in which in Ia Q is absent and R1 is X-Arg-Gly-Asp-Y;
in Ib Q is -NH-(CH2)5-CO- and Rl is X-Arg-Gly-Asp-Y;
in Ic Q is -NH-(CHi)5-CO- and R1 is cyclo-(Arg-Gly-Asp-Z);
in Id Q is -NH-(CHz)5-CO- and R1 is cyclo-(Arg-Gly-Asp-M);
in Ie Q is -NFi-(CH~)5-CO- and R1 is A-Cys (R2) -BD
and i.n If Q is -NH- (CH2) n-CO-, RI is X-Arg-Gly-Asp-Y and n is 1, 2, 3, 4, 5 or 6.
The compounds of the formula I, and the starting materials to prepare them, are moreover prepared by methods known per se, as described in the literature (for example in the standard works such as Houben-Weyl, Methoden der organischen Chemie [Methods of organic chemistry], Georg-Thieme-Verlag, Stuttgart), specifically under reaction conditions which are known and suitable for the said reactions. It is moreover possible to make use of variants which are known per se but which are not mentioned here in detail.
The starting materials can, if required, also be formed in situ so that they are not isolated from the reaction mixture but immediately converted further into the compounds of the formula I.

Compounds of the formula I can preferably be obtained by reacting compounds of the formula II with compounds of the formula III.

The compounds of the formula II and III are known as a rule. If they are unknown, they can be prepared by methods known per se.

The radical -CO-L is the compounds of the formula III is a preactivated carboxylic acid, preferably a carbonyl halide, symmetrical or mixed anhydride or an active ester. Radicals of this type for activating the carboxyl group in typical acylation reactions are described in the literature (for example in the standard Works such as Houben-Weyl, Methodea der organischea Chemie, Georg-Thieme-Verlag, Stuttgart).

Activated esters are preferably formed is situ, for example by adding HOBt or N-hydroxysuccinimide.

L is preferably H, C1, Br or -ON-succinimide.

The reaction takes place, as a rule, in as inert solvent in the presence of an acid-binding agent, preferably an organic base such as triethylamine, dimethylaniline, pyridine or quinoline or an excess of the carboxyl component of the formula III.

It may also be beneficial to add as alkali metal or alkaline earth metal hydroxide, carbonate or bicarbonate or another salt of a Weak acid of the alkali metals or alkaline earth metals, preferably of potassium, sodium, calcium or caesium.

The reaction time depends on the conditions used and is between a few minutes and 14 days, and the reaction temperature is between about -30 and 140, normally between -10 and 90, is particular between about 0 and about 70.

Examples of suitable inert solvents are hydro-carbons such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, tetrachloro-methane, chloroform or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl - y - 15 - 2185394 ether, diisopropyl ether, tetrahydrofuran (THF) or dioxane; glycol ethers such as ethylene glycol monomethyl or monoethyl ether (methylglycol or ethylglycol), ethylene glycol dimethyl ether (diglyme);
ketones such as acetone or butanone; amides such as acetamide, dimethylacetamide or dimethylfo~am~de (DMF); nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (DMSO); carbon disulfide;
carboxylic acids such as formic acid or acetic acid;
vitro compounds such as nitromethane or nitrobenzene;
esters such as ethyl acetate, water or mixtures of the solvents mentioned.
Compounds of -the formula I can furthermore be obtained by reacting compounds of the formula IV with compounds of the formula V. The starting compounds of the formula IV and Y are, as a rule, known. If they are not known, they can be prepared by methods known per se.
The radical -CO-L in the compounds of the formula V is a preactivated carboxylic acid, preferably a carbonyl halide, symmetrical or mixed anhydride or an active ester. Radicals of this type for activating the carboxyl group in typical acylation reactions are described is the literature (for example in the standard works such as Houben-weyl, Methoden der organischen Chemie, Georg-Thieme-Verlag, Stuttgart).
L is preferably C1, Br or -ON-succinimide.
Reaction of compounds of the formula IV with compounds of the formula V takes place under the same conditions relating to the reaction time, temperature and solvent as described for the reaction of the compounds of the formula II with compounds of the formula III. -Linear open-chain compounds of the formula I in which R1 is X-Arg-Gly-Asp-Y or A-Cys(R~)-B
can furthermore be obtained by coupling, in the last step of the solid-phase synthesis, biotin in the same cycle as a normal N-terminally protected amino acid as ' last component, and cleaving the biotin-pegtide off the resin under normal conditions.

The solid-phase synthesis, cleavage off and purification are carried out as described by A. Joaczyk and J. Meieahofer in Peptides, Proc. 8th Am. Pept.

Symp., Eds. 9. Hruby and D.H. Rich, Pierce Comp. III, p. 73-77 (1983) or in analogy to the techniques described fa Aagew. Chem, ~, 375-391 (199x).

Open-chain linear compounds of the formulae II

' and IV can moreover be prepared by conventional methods of amino acid and peptide synthesis as described, for example, is the standard works and patent applications mentioned, for example also by the solid-phase synthesis of Merrifield (B.F. Gysin and R.e. Merrifield, J. Am. Chem.. Soc. 9~, 310Z ff.

(1972) ) .

Cyclic compounds of the formula II and IV is which Rl is cyclo-(Arg-Gly-Asp-Z) can be prepared by cyclizing the linear compounds as described, for example, in D8 43 10 643 or is Houben-Weyl, l.c., Volume 15/II, pages 1 to 806 (1974).

The compounds of the formula I can furthermore be obtained by liberating them from their functional derivatives by solvolysis, is particular hydrolysis, or by hydrogenolysis.

Preferred starting materials for the solvolysis or hydrogeaolysis are those which comprise is place of one or more free amino and/or hydroxyl groups corres-ponding protected amino and/or hydroxyl groups, pre-ferably those which have is place of an H atom which is connected to as N atom an amino protective group, for example those which correspond to the formula I but comprise in place of an NH= group an NHR' group (in which R' is an amino protective group, for example HOC

or CHZ) .

Further preferred starting materials are those which have in place of the H atom of a hydroxyl group a hydroxyl protective group, for example those which correspond to the formula I but comprise in place of a v - 1~ - 2185394 hydroxyphenyl group an R"O-phenyl group (in which R" is a hydroxyl protective group).
It is also possible for several - identical or different - protected amino and/or hydroxyl groups to be present in the molecule of the starting material. If the protective groups which are present differ from one another, they can in many cases be cleaved off selectively.
The term °amino protective group° 3s generally known and refers to groups which are suitable for pro tecting (blocking) as amino group from chemical reactions but which can easily be removed after the required chemical reaction has been carried out else where in the molecule. Typical groups of this type are, in particular, uasubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino pro-tective groups are removed after the required reaction (or sequence of reactions), their nature sad size are not otherwise critical; however, those with 1-20, in particular 1-8 C atoms are preferred. The term "acyl group" is to be interpreted in its widest sense is con-nection with the present process. It includes acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulfoaic acids, and, in particular, alkoxycarbonyl, aryloxycarbonyl and, especially, aralkoxycarbonyl groups. Examples of acyl groups of these types are alkanoyl such as acetyl, pro-pionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl such as beazoyl or toluyl; aryloxyalkanoyl such as POA;
alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxy-carbonyl; aralkyloxycarbonyl such as CBZ
("carbobenzoxy°), 4-methoxybenzyloxycarbonyl, FMOC;
arylsulfonyl such as Mtr. Preferred amino protective groups are BOC and -Mtr, also CBZ, Fmoc, benzyl and acetyl.
The term °hydroxyl protective group" is likewise generally known and refers to groups which are suitable for protecting a hydroxyl group from chemical reactions - I8 - 2 ~ 85394 but which can easily be removed after the required chemical reaction has been carried out elsewhere in the molecule. Typical groups of this type are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups, as well as alkyl groups. The nature and size of the hydroxyl protective groups is not critical because they are removed again after the required chemical reaction or sequence of reactions; groups with 1-20, in particular 1-I0, C atoms are preferred.
Examples of hydroxyl protective groups include benzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, with benzyl and tert-butyl being particularly preferred. The COOH groups in aspartic acid and glutamic acid are preferably protected in the form of their tert-butyl esters (for example Asp(O But)).
The liberation of the compounds of the formula I
from their functional derivatives takes place, depend-ing on the protective group used, for example with strong acids, preferably with TFA or perchloric acid, but also with other strong inorganic acids such as hydrochloric acid or sulfuric acid, strong organic carboxylic acids such as trichloroacetic acid or sulfonic acids such as benzene- or p-toluenesulfonic acid. The presence of an additional inert solvent is possible but not always necessary. Suitable and pre-ferred inert solvents are organic, for example carboxylic acids such as acetic acid, ethers such as tetrahydrofuraa or dioxane, amides such as DMF, halo-genated hydrocarbons such as dichloromethane, also alcohols such as methanol, ethanol or isopropanol, and water. Also suitable are mixtures of the abovementioned solvents. TFA is preferably used is excess without addition of another solvent, perchloric acid in the form of a mixture of acetic acid and 70% perchloric acid in the ratio 9:1. The reaction temperatures for the cleavage are preferably between about = [sic] and about 50°, preferably between 15 and 30° (room temperature).

The groups BOC, OBut and Mtr can be cleaved off, for example, preferably with TFA in dichloromethane or with approximately 3 to 5N HC1 in dioxane at 15-30°, the FMOC group with an approximately 5 to 50% solution of dimethylamine. Diethylamine or piperidine in DMF at 15-30°.
The trityl group is employed to protect the amino acids histidine, asparagine, glutamine and cysteine. The cleavage off takes place, depending on the required final product, with TFA/10% thiophenol, in which case the trityl group is cleaved off all the amino acids mentioned, and when TFA/anisole or TFA/thioanisole is used, only the trityl group on His, Asn and Gln is cleaved off, whereas it remains on the Cys side chain.
Protective groups which can be removed by hydro-genolysis (for example CBZ or beazyl) can be cleaved off, for example, by treatment with hydrogen is the presence of a catalyst (for example of a noble metal catalyst such as palladium, preferably on a support such as carbon). Suitable solvents in this case are those indicated above, especially, for example, alcohols such as methanol or ethanol or amides such as DMF. The hydrogenolysis is, as a rule, carried out at temperatures between about 0 and 100° and under pressures between about 1 and 200 bar, preferably at 20-30° and 1-10 bar. Hydrogenolysis of the CBZ group takes place, for example, well oa 5 to 10% Pd/C is methanol or with ammomium [sic] formate (in place of hydrogen) on Pd/C in methanol/DMF at 20-30°.
A base of the formula I can be converted with an acid into the relevant acid addition salt, for example by reacting equivalent amounts of the base and the acid in an inert solvent such as ethanol and subsequently evaporating. Acids particularly suitable for this reaction are those which provide physiologically accep-table salts. Thus, it is possible to use inorganic acids, for example sulfuric acid, nitric acid, hydro-halic acids such as hydrochloric acid or hydrobromic .- ~ _ 20 _ 2 ~ 8394 acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, also organic acids, in particular ali-phatic, alicyclic, araliphatic, aromatic or hetero-cyclic monobasic or polybasic carboxylic, sulfonic or sulfuric acids, for example formic acid, acetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malefic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethane-sulfonic acid, ethanedisulfonic acid, 2-hydroxy-ethanesulfonic acid, benzenesulfonic acid, p-toluene-sulfonic acid, naphthalenemono- and -disulfonic acids, laurylsulfuric acid. Salts with physiologically unacceptable acids, for example picrates, can be used to isolate and/or purify the compounds of the formula I.
On the other hand, an acid of the formula I can be converted by reaction with a base into one of its physiologically acceptable metal or ammonium salt.
Particularly suitable salts in this connection are the sodium, potassium, magnesium, calcium and ammonium salts, also substituted ammonium salts, for example the dimethyl-, diethyl- or diisopropylammonium salts, mono-ethanol-, diethaaol- or diisopropylammoaium salts, cyclohexyl, dicyclohexylammonium salts, dibenzyl-ethylenediammonium salts, furthermore, for example, salts with arginine or lysine.
The invention furthermore relates to the use of the compounds of the formula I and/or their physio logically acceptable salts for producing pharmaceutical preparations, in particular by non-chemical means. For this purpose they can be converted into a suitable dosage form together with at least one solid, liquid and/or semiliquid vehicle or ancillary substance and, where appropriate, is combination With one or more other active substances.
The invention furthermore relates to pharmaceutical preparations comprising at least one 21 - z ~ 85394 compound of the formula I and/or one of its physiologically acceptable salts.
These preparations can be used as pharma ceuticals is human or veterinary medicine. Suitable vehicles are organic or inorganic substances which are suitable for enteral (for example oral), parenteral, topical administration or for administration in the form of an inhalation spray and which do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, petrolatum. Used for oral administration are, in particular, tablets, pills, coated tablets, capsules, powders, granules, syrups, solutions or drops, for rectal administration are suppositories, for parenteral administration are solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants, for topical administration are ointments, creams or dusting powders. The novel compounds can also be lyophilized and the resulting lyophilizates used, for example, to produce injection products. The indi-cated preparations can be sterilized and/or comprise ancillary substances such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts to influence the osmotic pressure, buffer substances, colorants, flavourings and/or several other active substances, for example one or more vitamins. The sprays which can be used for administration as inha-lation spray comprise the active substance either dissolved or suepeaded is a propellant gas or mixture of propellant gases (for example COs or chlorofluoro-carbona). In this case,, the active substance is pre-ferably used is micronized form, it being possible for one or more additional physiologically tolerated sol-vents to be present, for example ethanol. Inhalation solutions can be administered using conventional inhalers.

The compounds of the formula I and their physio-logically acceptable salts can be used as integrin inhibitors for controlling diseases, in particular pathologically angiogenic disorders, thromboses, myocardial infarct, coronary heart diseases, arterio-sclerosis, tumours, osteoporosis, inflammations and infections.
For this purpose, the substances according to the invention can be administered as a rule in analogy to other known peptides which are commercially avail able, but in particular in analogy to the compounds described in US-A-4 472 305, preferably in doses bet-ween about 0.05 and 500 mg, in particular between 0.05 and 100 mg, per dosage unit. The daily dose is pre-IS ferably between about 0.01 and 2 mg/kg of bodyweight.
The specific dose for each patient depends, however, on a wide variety of factors, for example on the activity of- the specific compound employed, oa the age, body-weight, general state of health, sex, on the diet, on the time and route of administration, on the rate of excretion, medicinal substance combination and severity of the particular disorder for which the therapy is applied. Parenteral administration is preferred.
The novel compounds of the formula I can furthermore be used in analytical biology and molecular biology. This entails utilization of the ability of the biotinyl radical to form a complex with the glycoprotein avidin.
Baes of the biotin-avidin complex are disclosed in E.A. Bayer and M. Wilchek in Methods of Biochemical Analysis ~ø, 1-45 (1980) (Lit. 1).
The novel compounds of the formula I can be used as integrin liganda for .producing columns for affinity chromatography to prepare pure iategrias. The complex of an avidin-derivatized support material, for example Sepharose, and the novel compounds of the formula I is formed by methods known per se, as described, for example, in Lit. 1.

For this reason, no further details of this method are given at this point, and reference is made to the corresponding literature, for example Lit. 1.
Suitable polymeric support materials are the polymeric solid phases which are known in peptide chemistry and preferably have hydrophilic properties, for example crosslinked polysaccharides such as cellu lose, Sepharoae or SephadexR, acrylamidea, polymers based oa polyethylene glycol or teatacular polymersR.
The novel compounds of the formula I can also be used as diagnostic markers for anti-biotin antibody reactions in ELISA-type assays and is FACS
(Fluorescence Activated Cell Sorter) analysis.
The use of antibiotin antibodies for detecting biotin is disclosed by M. Berger, Biochemistry ~, 2338-2342 (1975). The use of immunoglobulin IgG derivatized with biotin is an enzyme immunoassay (ELISA) is described by B. Holmkov-Nielsen et al. in Journal of Chromatography, 2.22, 225-233 (1984).
J. Gao and S.J. Shattil describe in J. Tmmuaol.
Methods 1$l, 55-64 (1995) an ELISA test which detects substances which inhibit iategria a=Ib(3==I activation. In this case, biotinylated fibrinogen is employed for detection.
The use of flow cytometry in clinical cyto-diagnosis is described by G. Schmitz and G. Rothe in DG
Kliniache Chemie Mitteiluagen 24 (1993) No. 1, page 1-14.
The compounds of the formula I can furthermore be employed in force field microscopy (atomic force microscopy AFM) to measure the strength of ligand-receptor interactions.
Ligand preferably means, acomplex of avidia and the novel compounds of the formula I.
Receptor preferably means an integrin receptor.
E.-L. Florin et al. describe measurements of adhesion forces between an avidin-functionalized force field microscope and biotinylated agarose in Science 415-417 (1994).

264'14-384 All temperatures are stated in °C hereinbefore and hereinafter. In the following examples ~usual working up" means: if necessary, water is added, if necessary, depending oa the constitution of the final product, the pH is adjusted to between 2 and 10, ex-traction is carried out with ethyl acetate or dichloro-methane, the organic phase is separated off, dried over sodium sulfate and evaporated, and purification is carried out by chromatography on silica gel and/or by crystallization. Rf values oa silica gel; mobile phase:
ethyl acetate/methanol 9:1.
RT = retention time (minutes) on HPLC in the following systems:
[A]
TM
Column: Nucleosil 7C18 250 x 4 mm Eluent A: 0.1% TFA in water Eluent B: 0.1% TFA in acetonitrile Flow rate: 1 ml/min Gradient: 20-50% B/30 min [H]
50-minute gradient 0-80% 2-propanol in water with 0.3%
TFA at 1 ml/min oa a LichrosorbR RP Select H (7 Ecm) 250 x 4 mm column [C]
TM
Column: Lichrospher (5 fcm) 100RP8 125 x 4 mm Eluent A: 0.01 M Na phosphate pH 7.0 Eluent B: 0.005 M Na phosphate pH 7.0/60% by volume 2-propanol Flow rate: 0.7 ml/min Gradient: 1-99% B/50 min.
Mass spectrometry (MS):EI (electron impact ionization) Mr .
FAH (Fast Atom Bombardment) (M+H)' ~. _ 25 _ ~ 18539 DMPP-resin represents 4-(2',4'-dimethoxyphenylhydroxy-methyl)phenoxy-resin [sic], a super acid-labile resin which permits the synthesis of side-chain protected peptides.
E~nle 1 0.6 g of Fmoc-Lys(Boc)-OH is dissolved in 100 ml of dichloromethane, 1.2 equivalents of DMPP-resin, 1.4 equivalents of HOBt and 1.4 equivalents of DCCI are added, and the mixture is stirred at room temperature for 12 hours. Removal of the solvent results in Fmoc-Lys(Boc)-DMPP-resin. In a peptide synthesizer, Fmoc-Pro-OH is condensed With H-Lys(BOC)-DMPP-resin [liberated from Fmoc-Lys(BOC)-DMPP-resin with piperidine/DMF (20%)] by employing a three-fold excess of the protected proline. The coupling is carried out with DCCI/HOBt at room temperature. Fmoc-Pro-Lys(BOC)-DMPP-resin is obtained. Analogously, subsequent cleavage off of the Fmoc protective group and consecutive couplings with Fmoc-Ser(BUt)-OH, Fmoc-Asp(OBut)-OH, E~oc-Gly-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Bit-OH under reaction conditions repeated for each coupling - Liberation of the a-amino group with piperidine/DMF (20%) - Washing with dimethylacetamide - Reaction with the Fmoc-amino acid or Bit-OH
Bit-Gly-Gly-Gly-Arg(Mtr)-Gly-Asp(OBut)-Ser(BUt)-Pro-Lys(BOC)-DMPP-resin.
The resin is washed With CF3S03H/CH=Clz/Ha0 to result in Bit-Gly-Gly-Gly-Arg(Mtr)-Gly-Asp(OBut)-Ser(But)-Pro-Lys(BOC)-OH.
The protective groups are cleaved off with 2N HC1 in dioxane, the solvent if removed, the residue is taken up in TFA/CHzClz and precipitated with EtzO, and then purification is carried out by RP-HPLC.
Bit-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH x 2 TFA;

_ 26 _ 2185394 RT [B] = 12.14 is obtained; FAB 1056.
E~lpe2 In analogy to Example 1, consecutive couplings of the DMPP-resin with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH
and Bit-OH result in:
Bit-Gly-Gly-Gly-Lys{BOC)-Thr(But)-Ala-Asp(OBut)-Cys(Trt)-Pro-DMPP-resin.
Cleavage off from -the resin, cleavage off of the protective groups and purification result in Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-OH x2 TFA;
RT [B] 27.6; FAB 1273.
The following are obtained analogously by condensing the DMPP-resin with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Ala-Ala-Asp-Cys(Trt3-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Arg(Mtr)-OH and Bit-OH:
Bit-Arg-Thr-Ala-Aap-Cys(Trt)-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH and Bit-OH:
Bit-Ser-Ala-Asp-Cys(Trt)-Pro-OH;

~ . ~ _ 2, _ 2185394 with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH, Fmoc-Gln(Trt)-OH and Bit-OH:
Bit-Gln-Ser-Ala-Asp-Cys(Trt)-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH, Fmoc-Glp-OH and Bit-OH:
Bit-Glp-Ser-Ala-Asp-Cys(Trt)-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH, Fmoc-Ile-OH and Bit-OH:
Bit-Ile-Ser-Ala-Gly-Cys(Trt)-Pro-OH;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH, Fmoc-Arg(Mtr)-OH and sit-ox:
Bit-Arg-Ser-Ala-Gly-Cys(Trt)-Pro-OH;
with Fmoc-Pro-oH, Fmoc-cys(Trt)-oH, FMOC-Asp(oBut)-ox, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH and Bit-OA:
Bit-Lys-Gly-Gly-Asp-Cys(Trt)-Pro-OH;
with Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-OH;
with Fmoc-Thr(BUt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Floc-Ala-OH aad Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiophenol;
Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
b) When the protective groups are cleaved off With TFA and 10% thioanisole:
Bit-AIa-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;

with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-ox, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-oH, Fmoc-Pro-ox, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH and Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiophenol;
Bit-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
b) When the protetive groups are cleaved off with TFA and 10% thioanisole:
Bit-Gly-Lys-Thr-Cys(Trt)-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
with FmOC-Gly-OH, FEIOC-Lys(BOC)-OH, FEIOC-His(Trt)-OH, FmoC-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, FmoC-Lys(BOC)-OH and Bit-OH:
a) When the protective groups are cleaved off with TFA and 10% thiophenol:
Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;
b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;
with FmoC-Thr(But)-OH, FmoC-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, FmoC-Lys(BOC)-OH and Bit-OA:
a) When the protective groups are cleaved off With TFA and 10% thiophenol:

. .. ~ 2185394 Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
with Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH aad Bit-OH:
a) when the protective grougs are cleaved off with TFA and 10% thiopheaol:
Bit-Gly-Lys-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH:
b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-ASn-Pro-His-Lys-Gly-OHp with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-oH, Fmoc-Lys(BOC3-oH, Fmoc-His(Trt)-oH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH and Bit-OH:
Bit-Thr-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
with Fmoc-Thr(But)-OH, Pmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asa(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH and Bit-OH:
Bit-Ala-Asp-Cys-Pro-Arg-Asa-Pro-His-Lys-Gly-Pro-Ala-Thr-OH;
with Floc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asa(Trt)-OH, Fmoc-Arg(Mtr).-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Floc-ASp(OBut)-OH, Fmoc-Ala-OH and Bit-OH:
Bit-Ala-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-OH;
with E'moc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OA, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Emoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Aep-Cys(Trt)-Pro-Arg-Asn-Pro-OH;
with Fmoc-Lys(BOC)-OH, ~noc-His(Trt)-OA, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, P'moc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, P'moc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-OH;
with Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, E'moc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, l~oc-Thr(But)-OH, Fmoc-Lys(BOC)-OH aad-Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-OH;
with Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-off ana sit-oH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-OH;
with Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoe-Pro-OH, Fmoc-Cys(Trt)-OH, Floc-Asp(OBut)-OH and Bit-OH:
Bit-Asp-Cys(Trt)-Pro-Arg-Asn-Pro-His-Lys-OH;
with Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH and Bit-OH:
Bit-Ala-Asp-Cys(Trt)-Pro-Arg-OH;

with Floc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(Hut)-OH, and Hit-OH:
Hit-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-OH;
with Fmoc-Pro-OH, F~noc-Cys (Trt) -OH, Fmoc-Asp (OHut) -OH, Fmoc-Ala-OH, Fmoc-Thr (But) -OH, Fbnoc-Lye (BOC) -OH and Bit-OH:
Hit-Lys-Thr-Ala-Aap-Cys(Trt)-Pro-OH;
with Pbnoc-NMeAla-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OHut)-OH, Fmoc-Ala-OH, Fmoc-Thr(8ut)-OH, F'moc-Lys(BOC)-OH and Hit-OH:
Hit-Lys-Thr-Ala-Asp-Cys(Trt)-NMeAla-OH;
Ex~
1.7 g of (+)-biotinyl-N-auccinimidyl ester, which is inexpensive, and 0.5 g of triethylamine are added to a aolutioa of 3.05 g of cyclo-(Arg-Gly 20. Asp-D-Phe-Lye) [obtainable by cyclizing 8-Arg(Mtr)-Gly Asp(OBut)-D-Phe-Lys(BOC)-OH to cyclo-(Arg(Mtr)-Gly-Asp(OHut)-D-Phe-Lys(BOC)) and subsequently cleaving off the protective groups] in 100 ml of dichloromethane.
The mixture is stirred at room temperature for 5 hours, and the usual working up results in cyclo-(Arg-Gly-Asp-D-Phe-Lys (NE-Hit) ) x TFA; RT [H] 11.32; FAB 830.
Example 4 In analogy to Example 3, cyclo-(Arg-Gly-Asp-D
Phe-Lys (N"-Hit-Aha) ) x TFA; RT [C] 23 .67; FAH 943, is obtained from 3.05 g of cyclo-(Arg-Gly-Asp-D-Phe-Lys) and 2.3 g of 'N-auccinimidyl (+)-biotinyl-6 aminocaproate (°A"), . which can be purchased inexpensively, and 0.5 g of triethylamine.
There are obtained analogously from "A" and the following cyclic compounds Cyclo-(Arg-Gly-Asp-D-Trp-Lys) Cyclo-(Arg-Gly-Asp-D-Tyr-Lys) - 32 - 2 i 85394 Cyclo-(Arg-Gly-Asp-D-Phe-D-Lys) Cyclo-(Arg-Gly-Asp-D-Phe-Cys) Cyclo-(Arg-Gly Asp-D-Phe-Dab) Cyclo-(Arg-Gly-Asp-D-Trp-D-Cys) Cyclo-(Arg-Gty-Asp-D-Tyr-D-Cys) Cyclo-(Arg-Gly-Asp-Phe-D-Lys) Cycto-(Arg-Gly Asp-Trp-D-Lys) Cyclo-{Arg-Gly-Asp Tyr-D-Lys) Cyclo-(Arg-Gly-Asp-Phe-D-Cys) Cyclo-(Arg-Gly-Asp-Phe-Dab) Cyclo-(Arg-Gly-Asp-Trp-D-Cys) Cyclo-(Arg-Gly-Asp-Tyr-D-Cys) Cyclo-(Arg-Gly-Asp-D-Trp-Om) Cyclo-{Arg-Gly-Asp-D-Tyr-Om) Cyclo-(Arg-Gly-Asp-D-Phe-Om) Cyclo-(Arg-Gly-Asp-D-Trp-D-Orn) Cyclo-(Arg-Gly-Asp-D-Tyr-D-Orn) Cycto-(Arg-Gly-Asp-D-Phe-D-Orn) Cyclo-(Arg-Gly-Asp-D-Trp-Dab) Cyclo-(Arg-Gly-Asp-D-Tyr-Dab) Cyclo-(Arg-Gty-Asp-D-Trp-Dap}
Cyclo-(Arg-GIyAsp-D-Tyr-Dap) Cyclo-(Arg-Gly-Asp-D-Phe-Dap}
Cyclo-(Arg-Gly-Asp-D-Trp-D-Dap) Cyclo-{Arg-Giy-Asp-D-Tyr-D-Dap) Gyclo-(Arg-Gfy-Asp-D-Phe-D-Dap) the following compounds Cyclo-{Arg-Gly-Asp-D-Trp-Lys(N'-Bit-Aha)) Cyclo-(Arg-Gty-Asp-D-Tyr-Lys(N'-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Phe-D-Lys(N'-Bit-Aha)) Cycto-(Arg-Gly Asp-D-Phe-Cys(S-Bit-Aha)) Cycto-(Arg-GIyAsp-D-Phe-Dab(N'-Bit-Aha)) Cycio-(Arg-Gly Asp-D-Trp-D-Cys(S-Bit Aha}) Cyclo-(Arg-G(y-Asp-D-Tyr-D-Cys(S-Bit Aha)) Cyclo-(Arg-Gly-Asp-Phe-D-Lys(N'-Bit-Aha)) Cyclo-(Arg-Gly-Asp-Trp-D-Lys(N'-Bit-Aha)) Cyclo-(Arg-Gly-Asp-Tyr-D-Lys{N'-Bit Aha)) Cyclo-(Rrg-Gly-Asp-Phe-D-Cys(S-Bit Aha)) Cyclo-(Arg-Gly Asp-Phe-Dab(NY-Bit-Aha)) Cyclo-(Arg-Gly-Asp-Tip-D-Cys(S-Bit Aha)) Cyclo-(Arg-Gly-Asp-Tyr-D-Cys(S-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Trp-Om(Na-Bit-Aha)) Cycio-(Arg-Gly Asp-D-Tyr-Om(Na-Bit Aha)) Cyclo-(Arg-Gly-Asp-D-Phe-Om(Na-Bit Aha)) Cyclo-(Arg-Gly-Asp-D-Trp-D-Om(Na-Bit Aha)) Cyclo-(Arg-Gly Asp-D-Tyr-D-Om(Na-Bit-Aha)) Cyclo-(Arg-Gty Asp-D-Phe-D-Om(Na-Bit-Aha)) Cyclo-(Arg-G!y-Asp-D-Trp-Dab(N'-Bit-Aha)) Cycio-{Arg-Gly-Asp-D-Tyr-Dab(N'-Bit-Aha)) Cyclo-{Arg-Gly-Asp-D-Trp-Dap(N°-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Tyr-Dap(Na-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Phe-Dap(N°-Bit-Aha)) Cycto-(Arg-Gly-Asp-D-Trp-D-Dap(N~-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Tyr-D-Dap(N°-Bit-Aha)) Cyclo-(Arg-Gly-Asp-D-Phe-D-Dap(Na-Bit-Aha)) E
6 g of Boc-Aha N-succinimidyl ester are added to a solution of 3.05 g of cyclo-(Arg-Gly-Asp-D-Phe-Lys) in 40 ml of 5$ aqueous NaHC03 and 40 ml of THF. Stirring for 4 hours and the usual working up result in cyclo-(Arg-Gly-Asp-D-Phe-Lys(BOC-Aha)); RT [C) 27.7; FAB 817.
Cleavage off of the BOC group in HC1/dioxane and the usual working up result is cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'-Aha)) x 2 TFA; RT [C] 14.76; FAB 717.
In analogy to Example 1, subsequent reaction with (+)-biotinyl-N-succinimidyl [sic? ester results in cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'-Bit-Aha)) x 2 TFA; RT
[C] 23.67; FAB 943.

,~ - 34 - 2185394 Exam, 1~
In analogy to Example 4 there is obtained from cyclo-(Arg-Gly-Asp-D-Phe-Lys-Gly) [obtainable by cyclizing H-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys(BOC)-Gly-S OH to cyclo-(Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Lys(BOC)-Gly) and subsequently cleaving off the protective groups]
and N-succinimidyl (+)-biotinyl-6-aminocaproate cyclo-(Arg-Gly-Asp-D-Phe-Lys(N'-Bit-Aha)-Gly) x TFA; RT
(A] 10.97; FAB 1000.
There is obtained analogously from cyclo-(Arg-Gly-Asp-D-Phe-Val-Lys) [obtainable by cycliziag H-Arg(Mtr)-Gly-Asp(OBut)-D-Phe-Val-Lys(BOC)-O8 to cyclo-(Arg(Mtr)-Gly-ASp(OBut)-D-Phe-Val-Lys(BOC)) and subsequently cleaving off the protective groups] and N-succinimidyl (+)-biotinyl-6-aminocaproate cyclo-(Arg-Gly-Asp-D-Phe-Val-Lys(N'-Bit-Aha)) x TFA; RT (A] 16.11;
FAB 1042.
There is obtained analogously from cyclo-(Arg-Gly-ASp-D-Phe-N-Ne-Lys) and N-succinimidyl (+)-biotinyl-6-aminocaproate cyclo-(Arg-Gly-Asp-D-Phe-N-Me-Lys(N'-Bit-Aha)).
ale 7 Ia analogy to Example l, successive couplings onto an MBHA-resin with the addition of 1.4 equivalents of HOBt and 1.4 equivalents of DCCI with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH and Bit-OH result ia:
Bit-Gly-Gly-Gly-Lys(BOC)-Thr(But)-Ala-Asp(OBut)-Cys(Trt)-Pro-MBSA-resin.
Cleavage off from the resin with TFA, cleavage off of the protective groups with piperidine/DMF and puri fication result is Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-NH, There are obtained analogously by condensation of the NBHA-resin 35 - 21 t35~94 with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-NH2:
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Ala-Ala-Asp-Cys(Trt)-Pro-NHa with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBUt)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Arg(Mtr)-OH
and Bit-OH:
Bit-Arg-Thr-Ala-Asp-Cys(Trt)-Pro-NH2 with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(08ut)-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH and Bit-OH:
Bit-Ser-Ala-Asp-Cys(Trt)-Pro-NH2s with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, L~'moc-Ala-OH, Fmoc-Ser(8ut)-OH, Fmoc-Gln(Trt)-OH, aad Bit-OH:
Bit-Gla-Ser-Ala-Asp-Cys(Trt)-Pro-NHz;
with Pmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Ser(But)-OH, Fmoc-Glp-OH, and Bit-OH:
Bit-Glp-Ser-Ala-Asp-Cys(Trt)-Pro-NHz:
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Pmoc-Ser(But)-OH, Fmoc-Ile-OH and Bit-OH:
Bit-Ile-Ser-Ala-Gly-Cys(Trt)-Pro-NHz;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-O$, Fmoc-Ser(8ut)-OH, Fmoc-Arg(Mtr)-OH
and Bit-OH:
Bit-Arg-Ser-Ala-Gly-Cys(Trt)-Pro-NH2:
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(08ut)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, and Bit-OH:
Bit-Lys-Gly-Gly-Asp-Cys(Trt)-Pro-DiHzs with Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-NHst with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(MtrD-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH and Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiophenol:
Bit-Ala-Asp-Cys-Pro-Arg-ASa-Pro-His-Lys-Gly-Pro-Ala-Thr-DTHs s b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Ala-Asp-Cys(Trt)-Pro-Arg-ASa-Pro-His-Lys-Gly-Pro-Ala-Thr-NHa;
with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Ditr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH and Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiophenol:
Bit-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-ASn-Pro-His-Lys-Gly-Pro-Ala-Thr-NHis b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Gly-Lys-Thr-Cys(Trt)-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-Lys-Gly-Pro-Ala-Thr-NH2s with Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-ASa(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Aap(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH
and Bit-OH:

' ~ - 3~ - 21 ~5~94 a) when the protective groups are cleaved off with TFA and 10% thiophenol:
Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-ASn-Pro-His-Lys-Gly-NHz ;
b) when the protective groups are cleaved off with TFA and 10% thioaaisole:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-Lys-Gly-NH=;
with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-ASa(Trt)-OH, FMoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-ASp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiophenol:
Bit-Lys-Thr-Ala-Asp-Cys-Pro-Arg-ASn-Pro-His-Lys-Gly-Pro-Ala-NHs;
b) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-ASa-Pro-His-Lys-Gly-Pro-Ala-Thr-IZHs;
with Pmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Pmoc-Cys(Trt)-OH, Fmoc-Asp(OBUt)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH, Fmoc-Gly-OH and Bit-OH:
a) when the protective groups are cleaved off with TFA and 10% thiopheaol:
Bit-Gly-Lys-Thr-Ala-Asp-Cys-Pro-Arg-ASn-Pro-His-Lys-Gly-NHs;
a) when the protective groups are cleaved off with TFA and 10% thioanisole:
Bit-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-Lys-Gly-NSs;

- 3$ - 218394 with Fmoc-Thr(But)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc Gly-OH, Fmoc-Lys(BOC)-OH. Fmoc-His(Trt)-OH, PSmoc-Pro OH, P'moc-ASa(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH. Fmoc Thr(But)-OH aad Bit-O8:
Bit-Thr-Ala-Asp-Cys-Pro-Arg-Asa-Pro-His-Lys-Gly-Pro-Ala-Thr-NHsp with Fmoc-Thr(BUt)-OH, Fmoc-Ala-OH, Rmoc-Pro-OH, Fmoc-Gly-OH. hoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH and Bit-OH:
Bat-Ala-Asp-Cys-Pro-Arg-Asa-Pro-His-Lys-Gly-Pro-Ala-Thr-NHat with Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH. Fmoc-His(Trt)-OH, Pmoc-Pro-OH, Pmoc-Asn(Trt)-OH, Fmoc-Arg(Bitr)-OH, Fmoc-Pro-OH. hoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH.
Fmoc-Ala-OH and Bit-08:
Bit-Ala-Asp-Cys-Pro-Arg-Asa-Pro-8is-Lys-Gly-NH2;
with Fmoc-Pro-OH, Fmoc-ASa(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBUt)-OH, Fmoc-Ala-OH. Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-NHs;
with Fmoc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asa(Trt)-OH, P'moc-Arg(Mtr)-OH, P'moc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Pmoc-Thr(BUt)-OH, Fmoc-Lys(BOC)-OH and Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-ASn-Pro-His-Lys-NHs:
with Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-ASa(Trt)-OH, Fmoc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH.
Fmoc-Lys(BOC)-OH aad Bit-OH:

Bit-Lys-Thr-AIa-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-NS~:
with Floc-Arg(D~tr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-O8, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(BOC)-O8 aad Hit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-NSz;
with P~oc-Lys(BOC)-OH, Fmoc-His(Trt)-OH, Pmoc-Pro-OH, Fmoc-Asa(Trt)-OH, Fmoc-Arg(8tr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-O8, Fmoc-Asp(OBut)-O8 and Bit-OH:
Bit-Asp-Cys(Trt)-Pro-Arg-Asa-Pro-His-Lys-NHz;
with Floc-Arg(Mtr)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(08ut)-OH, F'moc-Ala-OH aad Bit-OH:
Bit-Ala-Asp-Cys(Trt)-Pro-Arg-NBs;
with F'moc-Arg (8tr) -OH, Fmoc-Pro-OH, F'moc-Cys (Trt) -OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, F'moc-Thr(But)-08 aad Bit-OH:
Bit-Thr-Ala-Asp-Cys(Trt)-Pro-Arg-NHz;
with Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Ala-OH, Floc-Thr(But)-OH, Fmoc-Lys~(BOC)-OH aad Bit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-NBz;
with F'moc-NMeAla-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(08ut)-OH, Pmoc-Ala-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(80C)-08 aad Hit-OH:
Bit-Lys-Thr-Ala-Asp-Cys(Trt)-NMeAla-NHs;
Example 8 Production of a material suitable for affinity chromatography to purify iategrias:
The activatioa of Sepharose takes place as described is Lit. l, page 14. They 20 mg of avidia is 20 ml of 0.1 M sodium bicarbonate solution are added to TM
10 g of activated Sepharose.

- 40 - ~ ~ 85394 The suspension is stirred at 4° for 12 hours and then washed. The material is they suspended is water With a few crystals of sodium azide.
The avidia complex with the biotinylated compounds according to the invention, for example cyclo-(Arg-Gly Asp-D-Phe-Lys(N'-Bit)) x TFA, is formed by dissolving 1.1 equivalent of peptide in sodium acetate buffer, adding the solution to the suapeasioa of avidin Sepharose and stirring at 4° for 10 hours. Excess peptide is removed by washing.
The following examples relate to pharmaceutical preparations:
Example A: Vials A solution of 100 g of an active substance of the formula I and 5 g of disodium hydrogen phosphate is 3 1 of double-distilled water are (sic] adjusted to pH
6.5 with 2 N hydrochloric acid, sterilized by filtration, dispensed into vials and lyophilized and sealed under sterile conditions. Each vial comprises 5 mg of active substance.
Example B: Suppositories A mixture of 20 g of an active substance of the formula I with 100 g of soya lecithin and 1400 g of cocoa butter is melted, poured into moulds and left to cool. Each suppository comprises 20 mg of active substance.
Example C: Solution A solution is prepared from 1 g of as active substance of the formula I, 9.38 g of NaHaP04 - 2 H20, 28.48 g of NazHPO~ ~ 12 Ha0 and 0.1 g of benzalkoaium chloride is 940 ml of double-distilled water. The pH is adjusted to 6.8, the volume is made up to 1 1 and the solution is radiation-sterilized. This solution can be used in the form of eye drops.
Example D: Ointment .r - 41 - 2185394 500 mg of as active substance of the formula 3 are mixed With 99.5 g of petrolatum under aseptic conditions.
Example 8: Tablets A mixture of 1 kg of active substance of the formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is compressed to tablets is a conventional way so that 1D each tablet comprises 10 mg of active substance.
example F: Coated tablets Tablets are compressed is analogy to example 8 and are then provided in a conventional Way with a coating of sucrose, potato starch, talc, tragacanth and colorant.
example G: Capsules 2 kg of active substance of the formula I are packed into hard gelatin capsules in a conventional way so that each capsule comprises 20 mg of the active substance.
example H: Ampoules A solution of 1 kg of active substance of the formula I in 60 1 of double-distilled Water is sterilized by filtration, dispensed into ampoules and lyophilized and sealed under sterile conditions. each ampoule comprises 10 mg of active substance.
8xample I: Lahalation spray 14 g of active substance of the formula I are dissolved is 10 1 of isotonic NaCl solution, and the solution is used to fill commercial spray vessels with a pump mechanism. The solution can be sprayed into the mouth or nose. One puff (about 0.1 ml) corresponds to a dose of about 0.14 mg.

Claims

1. Bit-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro-Lys-OH; or a salt thereof.

2. Bit-Gly-Gly-Gly-Lys-Thr-Ala-Asp-Cys(Trt)-Pro-OH;
or a salt thereof.

3. cyclo-(Arg-Gly-Asp-D-Phe-Lys(N.epsilon.-Bit)); or a salt thereof.

4. cyclo-(Arg-Gly-Asp-D-Phe-Lys(N.epsilon.-Aha)); or a salt thereof.

5. cyclo- (Arg-Gly-Asp-D-Phe-Lys (N.epsilon.-Bit-Aha) -Gly) ; or a salt thereof.

6. cyclo-(Arg-Gly-Asp-D-Phe-Val-Lys(N.epsilon.-Bit-Aha)); or a salt thereof.

7. cyclo-(Arg-Gly-Asp-D-Phe-N-Me-Lys(N.epsilon.-Bit-Aha)); or a salt thereof.

8. cyclo-(Arg-Gly-Asp-D-Phe-Lys(N.epsilon.-Bit-Aha)); or a salt thereof.

9. cyclo-(Arg-Gly-Asp-D-Phe-Lys(BOC-Aha)); or a salt thereof.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 or a physiologically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

11. A pharmaceutical composition according to claim 10 for treatment of a pathologically angiogenic disorder, thrombosis, myocardial infarction, a coronary heart disease, arteriosclerosis, a tumour, osteoporosis, inflammation or an infection.

12. A pharmaceutical composition according to claim 10 for inhibition of an integrin.

13. A compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof for treatment of a pathologically angiogenic disorder, thrombosis, myocardial infarction, a coronary heart disease, arteriosclerosis, a tumour, osteoporosis, inflammation or an infection.

14. A compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof for inhibition of an integrin.

15. A use of a compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof for treatment of a pathologically angiogenic disorder, thrombosis, myocardial infarction, a coronary heart disease, arteriosclerosis, a tumour, osteoporosis, inflammation or an infection.

16. A use of a compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof for inhibition of an integrin.

17. A use of a compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof in manufacture of a medicament for treatment of a pathologically angiogenic disorder, thrombosis, myocardial infarction, a coronary heart disease, arteriosclerosis, a tumour, osteoporosis, inflammation or an infection.

18. A use of a compound according to any one of claims 1 to 9, or a physiologically acceptable salt thereof in manufacture of a medicament for inhibition of an integrin.

19. Use of a compound or salt according to any one of claims 1 to 9 for purifying an integrin by affinity chromatography.

20. Use of a compound or salt according to any one of claims 1 to 9 as a diagnostic marker for an anti-biotin antibody reaction in an ELISA-type assay and FACS analysis.

21. Use of a compound or salt according to any one of claims 1 to 9 in atomic force microscopy to measure the strength of a ligand-receptor interaction.